Con A Lab Report

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In the Affinity Chromatography experiment we were purifying our Con A proteins. In general, affinity chromatography is a technique that is used for isolating a protein, in our case Con A from a large amount of other macromolecules. Our protein of interest is captured using a microbead matrix while we let everything else flow through the column. The Sephadex matrix is made of cross-linked glucose or dextran and because our Con A has an affinity for glucose it is able to bind to those beads. In general, we began by equilibrating our column with NaCl, then poured Jack Bean Meal Extract which so happens to contain Con A through our column, the Con A then binded to the Sephadex beads, and finally we eluded with a dextrose solution so that
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We began the experiment by mixing our Con A with sheep red blood cells. If Con A is biologically active it is able to agglutinate the red blood cells. If Con A is inactive it results in no agglutination. Partial agglutination is another possible result. Our experimental variables were Con A + mannose and Con A + galactose. Ultimately we were comparing our purified Con A sample activity to a controlled Con A…show more content…
We found that Tube 1 had an absorbance of 0.085, Tube 2 had 0.034, Tube 3 had 0.027, Tube 4 had 0.032, Tube 5 had 0.025, Tube 6 had 0.028, Tube 7 had 0.022, Tube 8 had 0.024, Tube 9 had 0.030, and Tube 10 had an absorbance of 0.022. We then graphed absorbance vs fraction number so that we could demonstrate the absorbance of each tube and found that Tube 1 had our highest value of absorbance. Basically, the first few tubes have the most protein and then the rest drop off as is shown in the graph.

For the hemagglutination assay experiment the Con A concentration went from full strength at well A1 and at well A12 was the least. In the row of Con A by itself there was agglutination present in A1 through A6 but eventually the concentration became so dilute that around A7 the red blood cells were not able to agglutinate anymore. Similar results were seen with our Con A + galactose. Our Con A + mannose showed the first few wells with agglutination but eventually our Con A was not able to agglutinate the red blood cells due to the mannose interference. The negative control showed no agglutination throughout the wells. Our Con A wells should’ve been similar in comparison to the wells with control Con
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